Agonists often display marked differences in potency and efficacy for different responses despite interactions with a single receptor (sub)type. The applicants have shown that this is due to differences in receptor/effector coupling efficiency, assessed by receptor reserve (RR) analysis. This project will now investigate the underlying molecular basis of receptor coupling efficiency, focusing on preferential coupling of receptors with specific G proteins, using both in vivo and in vitro models derived from previous studies. Several observations form the basis for studies in the present proposal: 1) Subsensitivity (and reduction in RR) of 5-HT1A autoreceptors occurs after chronic treatment with partial, but not full, 5-HT1A agonists. This may be due to a differential ability of the agonists to alter receptor coupling to G proteins at pre- or postsynaptic loci; 2) the relative efficacies of agonists can be markedly altered for different responses, which suggests that different agonists may preferentially couple the same receptor to different G proteins; 3) nonselective reduction of G proteins (with pertussis toxin) alters agonist dose-response curves in a manner analogous to reduction of receptor density, indicating that the stoichiometric relationship between receptor and G protein governs the potency and efficacy of agonists. The following specific aims are proposed to test the hypotheses that: 1) The differential effects of partial (ipsapirone) and full (8-OH-DPAT) 5-HT1A agonists in eliciting 5-HT1A autoreceptor subsensitivity is due to: a) the ability of the latter but not the former drug to alter postsynaptic 5-HT1A receptors, G proteins or receptor-G protein coupling or b) these agonists preferentially couple to different G proteins which are differentially susceptible to regulation by chronic agonist treatment. Receptor autoradiography, immunoblotting and agonist- stimulated binding of [35S]GTPalphaS will be used to assess differential drug effects. 2) D2 receptors couple preferentially to Gi a3 to inhibit prolactin (PRL) release and stimulate K+ channel opening. A modified pituitary tumor-derived cell line (pRSVneo-transfected GH4C1) will be used in which the expression of D2S receptors regulating PRL secretion can be easily increased (by Zn+2) or decreased (by receptor alkylation). These cells will be transfected with vectors containing cDNA for pertussis toxin-sensitive G protein alpha subunits (Go, Gia2, Gia3) in both sense and antisense orientations, thereby increasing or decreasing the expression of individual alpha subunits. Dose-response curves for NPA inhibition of PRL release and second messenger responses (cAMP, CA+2 entry, K+ channel opening) in clones expressing various ratios of receptor and each alpha subunit will be generated. The relationship between receptor coupling efficiency and the identity and relative amount of alpha subunit, and density of D2 receptors, will be determined. 3) Different D2 receptor agonists couple preferentially to different G proteins. Dose-response curves for quinpirole and (+)3-PPP inhibition of prolactin secretion and second messenger responses will be generated in the above cell lines and the relative efficacies of the agonists will be determined for coupling with different ratios of G proteins. The results will aid in understanding factors subserving functional response at receptors (DA, 5-HT) involved in Parkinson's disease, schizophrenia and depression.